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Abstract

Neutralizing antibodies (Nabs) are a major challenge in clinical trials of adeno-associated virus (AAV) vector gene therapy, because Nabs are able to inhibit AAV transduction in patients. We have successfully isolated several novel Nab-escaped AAV chimeric capsids in mice by administrating a mixture of AAV shuffled library and patient serum. These AAV chimeric capsid mutants enhanced Nab evasion from patient serum with a high muscle transduction efficacy. In this protocol, we describe the procedures for selection of the Nab-escaped AAV chimeric capsid, including isolation and characterization of Nab-escaping AAV mutants in mice muscle.

Adeno-associated virus (AAV) vectors have been used in many preclinical studies and clinical trials. Many diseases have received eventual treatment using AAV gene therapy. However, the presence of neutralizing antibodies in circulation poses a major challenge for AAV vector application in future clinical trials. Many approaches have been explored to evade activities of Nab. Herein, we described the approach with directed evolution for selection of Nab-escaping mutants from an AAV shuffling library.

DNA shuffling is a powerful strategy for generating diverse mutants. Through successive rounds of phenotypic selection, DNA shuffling libraries were characterized by higher quality and more targeted diversification. High-throughput selection of capsid mutants from AAV shuffling libraries has been used as a promising strategy to explore AAV mutants with the abilities to target specific tissues and evade Nabs. However, most of these selecting methods were only tested in vitro; some studies even used rabbit anti-AAV2 sera or human intravenous immunoglobulin. The approach of in vivo selection of capsid mutants could provide a platform to generate more effective AAV mutants that not only escape Nab from patient serum but also enhance transduction in specific tissues.

The generation of a new AAV shuffling libraryNote: There are multiple methods that can be used to generate DNA shuffling library, and a handful of kits available from manufacturers. DNA shuffling library can be generated using JBS DNA shuffling kit. The process of generation of AAV shuffling library is shown in Figure 1.

Three days post-treatment, inject 107 virus particles (vg) of adenovirus (Adv) dl309 via i.m. into the same muscle to amplify AAV genomes in vivo. Note: Any replicable Adv can be used in this step.

Collect muscles from injected mice at 2 days post Adv administration, and extract total DNA using DNeasy Blood and Tissue Kit.Note: There are several methods that can be used to isolate DNA from tissues, and a handful of kits available from many manufacturers to do it. Here, we performed a genomic DNA isolation from mouse muscle using DNeasy Blood and Tissue Kit. In this protocol, the isolation of genomic DNA from muscle relies upon the columns and spin steps. Please follow the protocol of the manufacturer.

Amplify AAV mutant capsids by PCR assay using the genomic DNA from the muscle as templates. The sequences of the primers are F1 5’-CAACTCCATCACTAGGGGTTC and R1 5’-CATGGGAAAGGTGCCAGA, which are localized at the AAV2 rep and AAV2 ITR, respectively. Run PCR assay using PfuUltra High-Fidelity DNA polymerase, following the program in Table 2.

Table 2. PCR cycling parameters

Purify PCR products by the QIAquick PCR Purification Kit.

Digest with SwaI and XbaI and ligate into pXR2 digested with the same endonucleases. Transform the constructed plasmids into a culture of DH10b competent cells.Note: The plasmid pXR2 can be any expression plasmid with Rep gene of AAV for further AAV package.

Image luciferase expression in mice at 3 weeks post-injection. Anesthetize mice using an isoflurane vaporizer and inject with 120 mg/kg of D-Luciferin substrate intraperitoneally 5 min before imaging. Place the mice inside the camera box of the IVIS system. Start imaging with an exposure capturing in mice.

Data analysis

After the mixture of AAV shuffling library and patient serum was injected into the muscles of the mice, imaging was carried out by the IVIS Lumina In Vivo imaging system, and the photon signal was measured by Living Image software. The results were shown in Figure 1b of the original paper (Li et al., 2016).

The AAV mutant capsids were recovered and sequenced. The results were shown in Figure 1a of the original paper (Li et al., 2016).

The recovered AAV mutant capsids were used to package the AAV mutants. The mixtures of the AAV mutants and patient serum were administrated into muscles of mice. The imaging was carried out by the IVIS Lumina In Vivo imaging system and the photon signal was measured by Living Image software. The results were shown in Figure 2 of the original paper (Li et al., 2016).

This work was supported by NIH grants R01DK084033 (to C.L. and R.J.S.), P01HL112761, R01AI072176, R01AR064369 and U54AR056953 (to R.J.S). R.J.S. is the founder and a shareholder at Asklepios Biopharmaceutical. He holds patents that have been licensed by UNC to Asklepios Biopharmaceutical, for which he receives royalties. This protocol is adapted from Li et al. (2016).